WO2007134366A1 - EXTRACTION ET PURIFICATION D'α-AMYLASE - Google Patents

EXTRACTION ET PURIFICATION D'α-AMYLASE Download PDF

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Publication number
WO2007134366A1
WO2007134366A1 PCT/AU2007/000628 AU2007000628W WO2007134366A1 WO 2007134366 A1 WO2007134366 A1 WO 2007134366A1 AU 2007000628 W AU2007000628 W AU 2007000628W WO 2007134366 A1 WO2007134366 A1 WO 2007134366A1
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WO
WIPO (PCT)
Prior art keywords
extract
amylase
cell
process according
specific activity
Prior art date
Application number
PCT/AU2007/000628
Other languages
English (en)
Inventor
Michael Patane
Mitchell Parker
Akshat Talwalkar
Original Assignee
Protech Research Pty Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2006902753A external-priority patent/AU2006902753A0/en
Application filed by Protech Research Pty Ltd filed Critical Protech Research Pty Ltd
Publication of WO2007134366A1 publication Critical patent/WO2007134366A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2422—Alpha-amylase (3.2.1.1.) from plant source
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)

Definitions

  • the invention relates to extracting and purifying an enzyme from a cell, particularly, but not exclusively, to extracting and purifying ⁇ amylase Background of the invention ⁇ -amylase (EC 3.2.1.1), otherwise.
  • ⁇ -amylase EC 3.2.1.1
  • ⁇ -amylase acts on starch, glycogen and related polysaccharides including oligosaccharides in a random manner; reducing groups are liberated in the ⁇ - conformation.
  • ⁇ -amylase is particularly important in food industries for providing for a variety of polysaccharides and oligosaccharides.
  • ⁇ -amylase tend to be difficult to operate on a commercial scale, in terms of requiring sophisticated fermentation technology, including multiple steps for extraction, separation and purification as well as high capital investment. Some processes are characterised by an unacceptable loss in activity or wastage of ⁇ -amylase. Other processes can produce a crude or non purified final product that may have sub-optimal specific activity with some processes utilising Genetically Modified Organisms which require additional source identification.
  • the invention seeks to at least minimise one or more of the above identified problems or limitations and provides in certain embodiments, a process for purifying an ⁇ -amylase from a cell.
  • the process includes a step of heating an extract of a cell formed from a solution including at least one divalent cation, to increase the specific activity of a ⁇ - amylase in the extract.
  • the invention provides ⁇ -amylase produced by the process of the invention. In another aspect, the invention provides a cell including ⁇ -amylase produced by the process of the invention.
  • the divalent cation is Calcium.
  • the Calcium ions may be included in the extract in an amount to permit control of the denaturation of ⁇ -amylase when the extract is heated.
  • Calcium ions are included in the extract in an amount to at least limit the denaturation of ⁇ -amylase when the extract is heated.
  • the concentration of Calcium may be less than 100 mM.
  • a concentration of Calcium in a range between about 25 to 75 mM is particularly useful as further down stream processing of the extract for additional purification, such as ion exchange chromatography, may require substantial removal of Calcium, Accordingly a concentration of Calcium ions of about 50 mM is particularly useful.
  • the Calcium ions are provided in a concentration selected from the group consisting of 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM and 5OmM.
  • the solution may be buffered to about pH 7.0 using a suitable solution, such as Trisma base.
  • a process for purifying ⁇ - amylase from a cell includes a step of heating an extract of a cell formed from a solution including at least one divalent cation, to increase the specific activity of a ⁇ -amylase in the extract.
  • the solution has a pH of at least about 5.5, although higher ranges to about pH 9.0 are particularly useful for enhancing the specific activity of the enzyme in the extract.
  • a solution having a pH selected from the group consisting of 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 and 9.0 may be used.
  • a pH of 5.5 to 7.0 is preferred, however above this range the activity of the enzyme tends to be affected.
  • Sodium acetate or Trisma, buffered with HCl in a concentration of about 20OmM is particularly useful to provide the appropriate pH.
  • the constituents of an extract of a barley grain, shoot or rootlet include a number of enzymes and their inhibitors having activity for various carbohydrate and protein substrates.
  • the extract is typically heated to a temperature that permits removal of the inhibitors and denaturation of unwanted proteases, ancillary enzymes, or otherwise, destruction of activity of these enzymes in the extract.
  • temperatures less than 65 0 C are suitable for this purpose. It is particularly advantageous to heat the extract to between about 50 and 7O 0 C because at temperatures approaching 75 0 C and above, ⁇ -amylase activity may be lost, Accordingly, a temperature of about 65 0 C is particularly useful.
  • the extract is obtained from a barely cell
  • the barley cell may have been steeped and/or germinated prior to formation of the extract. Suitable steeping times are generally about 2 days although they may be longer or shorter. Suitable germination times are about 4 to 5 days, although may be longer or shorter. Steeping and germination may increase the expression or development of ⁇ - amylase in the cell before extraction.
  • a process for extracting and purifying ⁇ - amylase from a cell includes the following steps:
  • the extract is maintained in conditions for promoting the extraction and stabilization of the ⁇ -amylase in the extract for up to 5 hours prior to heating the extract.
  • the extract may be maintained at less than 1 O 0 C for less than 3 days, For example, the extract may be maintained between 0 to about 4 0 C for between about 1 to 48 hours.
  • ⁇ -amylase can be purified to virtual homogeneity from a cell extract by a process including the following steps:
  • step (c) utilising ultrafiltration and ion exchange chromatography to purify and concentrate ⁇ -amylase from the heat treated extract.
  • ⁇ -amylase can be further purified from a heat treated cell extract by tangential flow filtration. Accordingly, in certain embodiments, in step (c), ultrafiltration is utilised to purify ⁇ -amylase from the heat treated extract.
  • ⁇ -amylase can be further purified from a heat treated cell extract by cation exchange chromatography. Accordingly, typically, in step (c), cation exchange chromatography is utilised to purify ⁇ -amylase from the heat treated extract.
  • the extract is derived from a barley cell and is typically produced by milling or homogenising barley rootlets in an appropriate buffer.
  • a blender such as a Waring blender.
  • the extract may be produced by milling germinated barley grains using a roller mill following a predefined steeping and germination protocol.
  • the solution into which the ⁇ -amylase from the cell is released to form an extract is typically a buffered solution with a controlled pH.
  • Solutions prepared from Trisma base or sodium acetate are examples of such solutions.
  • a solution having a concentration of no more than about 300 mM Tris is suitable, for example, 200 mM Tris-HCL is particularly advantageous adjusted and maintained at a pH 7.0.
  • the processes of the invention are useful for purifying ⁇ -amylase from cells other than barley cells.
  • Other examples include cells of grains such as com, legumes, rice and wheat, and other vegetable matter.
  • processes of the invention are useful for isolating barley ⁇ -amylase from cells that contain a recombinant nucleic acid molecule that encodes barley ⁇ amylase.
  • examples of such cells include bacterial cells and yeast cells,
  • the process is particularly useful for modifying the oligo and polysaccharide content of an ingredient for use in the manufacture of foods and beverages. Accordingly, in one embodiment the process includes: (a) heating an extract of a cell formed from a solution including at least one divalent cation, to increase the specific activity of a ⁇ -amylase in the extract;
  • the carbohydrate -containing ingredient may be a dough for use in the production of breads and bread-like products, a wort for use in the production of beer and other alcohol containing beverages; a sugar mixture for use in the production of confectionary.
  • a process for producing a detergent including: (a) heating an extract of a cell formed from a solution including at least one divalent cation, to increase the specific activity of a ⁇ -amylase in the extract;
  • Example 1 Materials and equipment. Barley grains (Schooner variety) were obtained from Barrett Burston Malting,
  • Barley grains were steeped and germinated for a total of six days then milled at 400 rpm on a Kustnel Freres & Cie roller mill at a feed rate of 2 kg per minute with a gap setting of lmm using smooth rollers to crack the grains allowing extraction of the enzymes.
  • Milled grains (100kg) were then immersed in buffer (1501trs) containing 0.2M Tris-HCL (pH 7.0) and 5OmM Calcium Chloride and agitated in a D-Tank using counter directional paddles for 5 hours at a temperature of 10 0 C to extract the enzyme.
  • This crude enzyme extract was then filtered through the backed bed of grains, screened and centrifuged in a Sharpies centrifuge at 16,000 rpm at 1O 0 C at a feed rate of 120 Ltrs per hour to remove any precipitate.
  • the extract was then heat processed to 65 0 C and recirculated for one hour in a
  • SWEP plate heat exchanger to remove enzyme inhibitors and denature unwanted proteins and enzymes before being rapidly cooled to ⁇ 10°C by recirculation through a fabricated tubular heat exchanger immersed in a refrigerated glycol bath to avoid inactivation of the extracted ⁇ -amylase.
  • the heat treated extract was then re-centrifuged at 16,000rpm at 10°C to remove any flocculated proteins.
  • the heat treated and cooled extract was concentrated and buffer exchanged with
  • the buffer used for FPLC preparative gel filtration and cation exchange chromatography was 2OmM Sodium Acetate (pH 4.8) containing 1 OmM Calcium Chloride.
  • the eluent buffer for cation exchange chromatography used 0.3 M Sodium Acetate (pH 4.8) containing 1 OmM Calcium Chloride. All chromatography was conducted on an Amersham Pharmacia AICAT gradient processing FPLC system complete with a 900 model monitor, lamp and detector (set at 280nm), utilising a 920 model pump and Frac 950 fraction collector interfaced to a Compaq Deskpro Pentium III computer supporting Unicom analytical software.
  • Isolation of ⁇ -amylase was identified by protein bands on native electrophoresis gels and individual absorption peaks on cation exchange chromatography.
  • An LW Scientific UV- Visible spectrophotometer was used to measure enzyme activity operating at 400 nm. The system was controlled by a Celeron processor computer supporting LW Scientific Graphite version 3.1 analytical software.
  • Example 2 Preparation of the Alpha Amylase HR reagent substrate to calculate ⁇ -amylase activity.
  • the analysis of ⁇ -amylase provides for the use of a modified Megazyme Ceralpha
  • Method utilising a substrate containing blocked /?-nitro ⁇ henyl maltoheptaosidc (BPNPG7, 54.5mg) and thermostable ⁇ -glucosidase (125U at pH 6.0) prepared from a freeze dried mixture. Preparation involved dissolving the contents of one vial in 1 OmL of distilled water and using 0.2mL per assay with the remaining volume divided in ImL aliquots and stored frozen between uses.
  • BPNPG7 blocked /?-nitro ⁇ henyl maltoheptaosidc
  • thermostable ⁇ -glucosidase 125U at pH 6.0
  • the basis of the Ceralpha assay requires the extracted ⁇ -amylase to cleave glycosidic bonds within the blocked /j-nitrophenyl oligosaccharide substrate to yield p-nitrophenyl maltosaccharide with the thermostable a- glucosidase present in the substrate producing free glucose and /?-nitrophenol over a 10 min incubation at 4O 0 C.
  • the reaction is stopped by the addition of 3mL of a 1% (w/v) Trisma solution (pH 11.0) with absorbance read at 400nm.
  • Example 3 Preparation of a standard curve for protein to determine ⁇ -amylase specific activity.
  • Protein was determined using the BioRad micro assay procedure derived from the original method of Bradford utilising a standard curve produced for bovine serum albumin. Each analysis was conducted in duplicate requiring incubation at room temperature for 10 minutes with the absorbance measured at 595 ran. Standards were prepared in the range of 0.2 to 1.4 mg/mL of protein.
  • Example 4 Modified Ceralpha Method for the assay for ⁇ -amylase activity.
  • the assay requires O.lmL of the extracted enzyme solution to be diluted with 0.9 mL of 0.1 M Sodium Acetate buffer (pH 5.5) containing 5OmM Calcium Chloride pre- equilibrated at 4O 0 C for 5 minutes. From this solution, a 0.2mL enzyme aliquot is mixed with 0.2mL of the prepared Ceralpha substrate containing the blocked p-nitrophenyl maltoheptaoside and thermostable ⁇ -glucosidase which is incubated at 4O 0 C for 10 minutes.
  • a reference blank is prepared by adding 3mL of 1 % (w/v) Trisma to 0.2mL Ceralpha substrate and 0.2mL of distilled water.
  • Example 5 Preparation of the crude ⁇ -amylase extract.
  • the germinated grains were then milled using counter rotating smooth rollers at a roller speed 400 rpm and gap setting of lmm with a feed rate of 2kg of grain per minute.
  • Extraction of ⁇ -amylase was conducted by immersion of the milled grains in a buffered solution of 0.2M Tris-HCl ( ⁇ H7.0) containing 5OmM CaCl 2 for 5 hours at 1O 0 C to facilitate solubilization of ⁇ -amylase.
  • the insoluble material was removed by screening the extract through compacted grains in the tank and then through a 10 micron filter. The filtrate was then centrifuged at 16,000 rpm at 1O 0 C to remove any remaining solids and then stored at 4 0 C until required. This process formed the crude ⁇ amylase extract.
  • Example 6 Purification of ⁇ -amylase from the crude extract.
  • the first stage of the purification process involved the removal of heat labile proteases, inhibitory proteins and any superfluous proteinaceous materials from the crude ⁇ -amylase extract with the aim of reducing any loss of activity or damage to the structure of ⁇ -amylase while increasing the specific activity of the ⁇ -amylase extract.
  • the crude extract was heated in plate heat exchanger to 65 0 C and maintained at that temperature for 1 hour.
  • the extract was then cooled in a tubular heat exchanger to ⁇ 10°C and centrifuged to remove the denatured proteinaceous materials.
  • the post heated enzyme extract was then concentrated, purified and buffer exchanged by cross flow ultrafiltration with 2OmM Sodium Acetate (pH 4.8) to facilitate down stream cation exchange chromatography.
  • Cation exchange chromatography was conducted using a 0.45 micron filtered series of 50 mL samples of the heated treated and cooled extract injected into a Super loop 50 column and run through a pre-equilibrated 30ml carboxymethyl cellulose cation exchange column at a flow rate of 2.0 mL per minute to fractionate ⁇ -amylase.
  • the isolated fractions were concentrated on a MidGee 1 OkDa TFF ultrafiltration unit combining a Masterflex economy drive peristaltic pump and Masterflex Easy load II head at a flow rate of 15 mL per minute.
  • a predominant single peak for ⁇ -amylase was obtained and analysed for activity and specific activity according to Examples 2, 3 and 4 above.
  • Example 7 Purification profile for ⁇ amylase. Results for the purification of ⁇ amylase are shown in Table 1. Table 1
  • Example 8 Extraction of ⁇ -amylase over an extended germination period.
  • Chloride to promote and stabilise the extracted enzyme at a moisture content not exceeding 40.0% (w/w). Grains were then germinated for a period of 1 to 5 days with a sample of the grains milled each day and the specific activity determined. The most effective time recorded for an increase in enzyme extraction based on the enzyme specific activity was seen at day 6 (2 days steep and 4 days germination) [84.19 ⁇ moles/min/mgj compared to day 3 (2 days steep and 1 day germination) [18.29 ⁇ molcs/min/mg] providing a 4.6 fold increase in specific activity declining at day 7 [75.03 ⁇ moles/min/mg].
  • Example 9 Germination profile for ⁇ amylase.
  • Example 10 Effect of extraction time on the activity of ⁇ amylase.
  • Example 11 Extraction time profile for ⁇ -amylase.
  • Example 12 Effect of pH on the activity of ⁇ amylase.
  • the specific activity was seen to increase from 7.10 ⁇ moles/min/mg at pH 9.0 to 98.01 ⁇ moles/min/mg at pH 7.0.
  • Example 13 Activity profile for g-amylase over a pH gradient.
  • Example 14 Effect of cations on ⁇ -amylase activity .

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Abstract

L'invention concerne un procédé d'extraction et de purification d'une α-amylase à partir d'une cellule. Ledit procédé consiste à chauffer l'extrait cellulaire en présence d'au moins un cation divalent afin d'augmenter l'activité spécifique de l'α- amylase. L'invention concerne également les utilisations d'une enzyme ainsi préparée dans la production ou la modification de produits alimentaires, de boissons et de détergents.
PCT/AU2007/000628 2006-05-22 2007-05-10 EXTRACTION ET PURIFICATION D'α-AMYLASE WO2007134366A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2006902753A AU2006902753A0 (en) 2006-05-22 Extracting and purifying alpha amylases
AU2006902753 2006-05-22

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WO2007134366A1 true WO2007134366A1 (fr) 2007-11-29

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3776693A (en) * 1972-01-24 1973-12-04 Dow Chemical Co Dry cleaning composition and process
JPS51106786A (en) * 1975-03-14 1976-09-21 Mitsubishi Petrochemical Co Tainetsuseiarufua amiraazeno seizoho
DD289287A5 (de) * 1989-11-23 1991-04-25 Adw,Zentralinstitut Fuer Ernaehrung, Rehbruecke,De Verfahren zur isolierung von keimungsspezifischer alpha-amylase aus getreide
WO1996023873A1 (fr) * 1995-02-03 1996-08-08 Novo Nordisk A/S Alleles d'amylase-alpha
EP0860500B1 (fr) * 1997-02-21 2004-09-01 Cerestar Holding B.V. Alpha amylase purifiée d'origine fongique stable en milieu acide

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3776693A (en) * 1972-01-24 1973-12-04 Dow Chemical Co Dry cleaning composition and process
JPS51106786A (en) * 1975-03-14 1976-09-21 Mitsubishi Petrochemical Co Tainetsuseiarufua amiraazeno seizoho
DD289287A5 (de) * 1989-11-23 1991-04-25 Adw,Zentralinstitut Fuer Ernaehrung, Rehbruecke,De Verfahren zur isolierung von keimungsspezifischer alpha-amylase aus getreide
WO1996023873A1 (fr) * 1995-02-03 1996-08-08 Novo Nordisk A/S Alleles d'amylase-alpha
EP0860500B1 (fr) * 1997-02-21 2004-09-01 Cerestar Holding B.V. Alpha amylase purifiée d'origine fongique stable en milieu acide

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Week 199138, Derwent World Patents Index; Class D16, AN 1991-274422, XP008097740 *
FERNANDEZ-TARRAGO J. ET AL.: "Purification and characterization of an alpha-amylase from the cotyledons of germinating lentils", REV. ESP. FISIOL., vol. 37, 1981, pages 197 - 204, XP008090991 *
KOCHHAR S. ET AL.: "Thermostable liquefying alpha-amylase from Bacillus amyloliquefaciens", BIOTECHNOL. LETTS., vol. 12, no. 5, 1990, pages 393 - 396, XP008090501 *
KRISHNAN T. ET AL.: "Purification and characterization of alpha-amylase from Bacillus licheniformis CUMC305", APPL. ENVIRON. MICROBIOL., vol. 46, no. 2, 1983, pages 430 - 437, XP008091076 *
PALACIOS H.R. ET AL.: "Effect of alpha-amylases from different sources on the retrogradation and recrystallization of concentrated wheat starch gels: relationship to bread staling", J. AGRIC. FOOD CHEM., vol. 52, no. 19, 2004, pages 5978 - 5986, XP008090871 *
PALACIOS H.R. ET AL.: "Effects of alpha-amylases from different sources on the firming of concentrated wheat starch gels: relationship to bread staling", J. AGRIC. FOOD CHEM., vol. 52, no. 19, 2004, pages 5987 - 5994, XP008090870 *
SHAW J.-F. ET AL.: "Studies on the alpha-amylase from the germinated rice seeds", BOT. BULL. ACADEMIA SINICA, vol. 23, 1982, pages 45 - 61, XP008090581 *
SYNOWIECKI J. ET AL.: "Sources, properties and suitability of new thermostable enzymes in food processing", CRIT. REV. FOOD SCI. NUTR., vol. 46, no. 3, April 2006 (2006-04-01), pages 197 - 205, XP008090716 *

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